Citric acid (2-hydroxy-propane-1,2,3-tricarboxylic acid) is known as an industrially important organic acid which is used e.g. as food additive, preservative or as stabilizator of oils and fats due to its ability to complex heavy metal ions like copper and iron. Originally, it has been isolated from citrus plants. Chemical synthesis of citric acid is also possible, however, not at all suitable for industrial production due to the expensive raw materials and a complicated process with low yield.
Therefore, over the past decades, other approaches to manufacture citric acid using microbial conversions, which would be more economical as well as ecological, have been investigated.
Citric acid production from a number of substrates including glucose or sucrose has been reported in several microorganisms, such as fungi including yeasts, using different cultivation methods. Examples of known fungi able to directly produce citric acid include, for instance, strains from the genera of Aspergillus, in particular A. niger, or yeasts such as Yarrowia, in particular Yarrowia lipolytica. 
The conversion of a substrate e.g. carbohydrates, into citric acid may involve many different metabolic routes, and involve several enzymatic steps to generate citric acid. Furthermore, transporters may also play an important role in the efficient conversion of a substrate into citric acid.
Proteins, in particular transporters, that are active in the transport of substances such as carbohydrates like e.g. glucose or sugar alcohols, carboxylates, minerals, toxic compounds like reactive oxygen, and related compounds over a membrane are herein referred to as being involved in the Transport System. This transport can be into the cytosol, into/out of a mitochondrion, vacuole, endoplasmatic reticulum, peroxisome or across another membrane barrier. Such proteins are abbreviated herein as TS proteins and function in the synthesis of citric acid or have a function in the cellular process of citric acid synthesis.
TS proteins are in general membrane-bound or are associated to membrane-bound structures and are functional as single proteins or as subunits in protein complexes such as permeases or active transporters. TS proteins are known to be responsible for selectively facilitating, assisting or enabling the transport of compounds such as sugars, sugar alcohols, carboxylates, minerals, toxic compounds across the cellular, periplasmatic or mitochondrial vacuolar, endoplasmatic reticulum or peroxisomal membrane.
TS proteins can be divided into several types on the basis of their mechanisms. The first class of transporters, also called ion channels, uses energy from the proton-motive force to transport molecules against a concentration gradient. These symport and antiport systems couple the movement of two different molecules across the membrane (via permeases having two separate binding sites for the two different molecules); in symport, both molecules are transported in the same direction, while in antiport, one molecule is imported while the other is exported.
A further class of transporters, also called “secondary transporters”, the phosphotransferase system (PTS), is energized by the transfer of a high-energy phosphate group from phosphoenolpyruvate, through various protein components, to the substrate which is phosphorylated upon import as its phosphoester form. Ion transporters of this group facilitate diffusion over a membrane, such as for example the cation diffusion facilitator (CDF) family or exchange anions such as for example the anion exchanger (AE) family. The group of major facilitator superfamily (MFS) contains many important sugar transporters.
A further class of transporters couples the hydrolysis of ATP to substrate translocation. These systems are termed the ATP-binding cassette (ABC) type transporters. The ABC-transport system consists of a substrate specific binding protein which is located in the periplasm in gram-negative bacteria or which is membrane associated in gram-positive bacteria, an integral membrane domain and a cytoplasmic-facing ATP hydrolyzing domain.
A more detailed description of membrane transport systems can be found in: Bamberg, E. et al (1993) “Charge transport of ion pumps on lipid bilayer membranes”, Q. Rev. Biophys. 26:1-25; Findlay, J. B. C. (1991) “Structure and function of membrane transport systems”, Curr. Opin. Struct. Biol. 1:804-810; Higgins, C. F. (1992) “ABC transporters from microorganism to man” Ann. Rev. Cell Biol. 8:67-113; Gennis, R. B. (1989) “Pores, channels and transporters” in: Biomembranes, Molecular Structure and Function, Springer, Heidelberg, p. 270-322; Nikaido, H. & Saier H. (1992) “Transport proteins in bacteria: common themes in their design” Science 258:936-942.
Preferably, the TS proteins or subunits of such proteins having activity towards or which are involved in the synthesis of citric acid from a carbohydrate are selected from the group consisting of hexose transporters, ion transporters, kinases, permeases, symporters, antiporters, mitochondrial carriers such as citrate transport proteins or tricarboxylate carriers, suppressors for mitochondrial histones, and metal transporters such as Manganese transporters or manganese resistance protein or iron transporters.
Proteins, in particular enzymes, that are involved in the citrate synthesis such as enzymes which take part in the TCA, like e.g. enzymes catalyzing the condensation of acetyl-CoA with oxaloacetate to form citric acid, are herein referred to as being involved in the Citrate-synthesis System and abbreviated as CS proteins which function in the synthesis of citric acid.
Preferably, the CS proteins or subunits of such proteins having activity towards or which are involved in citrate synthesis are selected from the group consisting of citrate synthases, glyoxysomal citrate synthases, aconitases, aconitate hydratases or hydrolylases, and 6-phosphofructokinases.
Proteins, in particular enzymes which are involved in side-reactions such as e.g. Mn-dependent mitochondrial superoxide dismutase (MnSOD), genes that are involved in so-called “by-pass routes” of the synthesis pathway of citric such as e.g. oxaloacetate hydrolases, glucose oxidase and/or glycolate (oxido-)reductases acid may have an influence in the cellular process of citric acid synthesis. Such enzymes are abbreviated herein as BS proteins or BS enzymes.
Production of citric acid using strains of Aspergillus has been reported previously (see, e.g. Karaffa and Kubicek, Appl Microbiol Biotechnol, 61:169-196, 2003). However, the yields and or productivity of citric acid production as known in the prior art may still be improved, which is an object of the present invention.